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Endocrinology Vol. 138, No. 6 2530-2536
Copyright © 1997 by The Endocrine Society

ARTICLES

Angiotensin-Responsive Adrenal Glomerulosa Cell Proteins: Characterization by Protease Mapping, Species Comparison, and Specific Angiotensin Receptor Antagonists1

Mary E. Elliott, Theodore L. Goodfriend, Dennis L. Ball and Colin R. Jefcoate

William S. Middleton Memorial Veterans Hospital and the Departments of Medicine and Pharmacology, University of Wisconsin School of Medicine, Madison, Wisconsin 53705

Address all correspondence and requests for reprints to: Mary E. Elliott, Ph.D., Hypertension Research Laboratory, Room C4114, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (AngII)-stimulated aldosterone synthesis is mediated by the AngII type 1 (AT1) receptor and requires ongoing protein synthesis. Hormonally-stimulated turnover of a family of 28- to 30-kDa proteins (p30, or steroidogenic acute regulatory proteins) has been linked to enhanced steroid synthesis in several tissues. Our previous work showed that AngII, dibutyryl cAMP, potassium, and atrial natriuretic peptide affected labeling of a group of eight proteins (four of 28 kDa and four of 30 kDa) in bovine adrenal glomerulosa cells. This report extends our findings in three ways: 1) The eight [35S]-methionine-labeled p30 proteins in bovine cells were compared with each other by chymotryptic peptide mapping. Similarity in maps indicated that the eight proteins share a common primary structure. 2) Dibutyryl cAMP treatment of rat adrenal glomerulosa cells affected the levels of four 28-kDa proteins and one 35-kDa protein, whereas AngII affected two of the 28-kDa proteins. There were no responsive 30-kDa proteins in rats comparable with those seen in bovine cells. These results indicate a species difference in the affected proteins. 3) The AT1 receptor antagonist, losartan, inhibited the effects of AngII on aldosterone synthesis and turnover of the p30 proteins in bovine adrenal glomerulosa cells. PD123319, an antagonist specific for the AngII type 2 receptor, did not block AngII-stimulated aldosterone synthesis and had much less effect on p30 protein labeling than did losartan. These results add to the growing body of evidence that this family of p30 or steroidogenic acute regulatory proteins plays a role in the acute regulation of steroidogenesis by a wide variety of stimulatory hormones in several tissues and species. In addition, losartan’s inhibition of AngII’s effects on the p30 proteins is consistent with a key role for these proteins in processes linking occupation of the AT1 receptor to stimulation of aldosterone synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STIMULATION of aldosterone synthesis by angiotensin II (AngII) is mediated by the AngII type 1 (AT1) receptor. AT1 receptor occupancy triggers hydrolysis of phosphatidylinositol 4,5-bisphosphate with subsequent increases in intracellular inositol 1,4,5-trisphosphate, diacylglycerol, and calcium (1, 2, 3, 4, 5, 6, 7, 8). Protein phosphorylation by calcium/calmodulin-stimulated protein kinase and protein kinase C leads to increased aldosterone synthesis. AngII’s stimulation of aldosterone synthesis shares key features with ACTH (cAMP) activation of steroid synthesis (9, 10, 11, 12, 13, 14, 15, 16). AngII and cAMP acutely increase conversion of endogenous cholesterol to pregnenolone by the mitochondrial enzyme cytochrome P450SCC by increasing the transfer of cholesterol from the outer to inner mitochondrial membrane where P450SCC is located. Protein synthesis is required for delivery of cholesterol to P450SCC (12, 13), and steroidogenesis is blocked by protein synthesis inhibitors.

A labile protein has been postulated to mediate the effect of ACTH (17). Orme-Johnson and colleagues have described a 28- to 30-kDa protein (pp30) that has the characteristics expected of such a protein (18, 19, 20, 21, 22, 23, 24, 25). Pp30 is found in the inner mitochondrial membrane, consistent with a role to increase mitochondrial use of cholesterol (23). Protease mapping and other studies indicated that pp30 is a phosphorylated form of another protein (p30) found in nonstimulated adrenal cells and that pp30 is made from a 37-kDa precursor, pp37 (24). Recent studies of the signal transduction pathways in bovine and rat adrenal tissue have confirmed that cAMP-dependent phosphorylation of pp37 protein occurs in both species but that protein kinase C stimulates pp37 phosphorylation in bovine, but not in rat, adrenal cells (25).

Mitochondrial proteins related to the pp30 adrenal proteins also are stimulated by HCG or LH in MA-10 mouse Leydig tumor cells (26, 27, 28, 29, 30, 31, 32, 33). Protease mapping indicates that they constitute a family made from one core protein and differing by posttranslational modification. The gene for this protein, named StAR (steroidogenic acute regulatory protein), has been cloned (31), and a human deficiency in steroid synthesis (congenital adrenal lipoid hypoplasia) has been linked to mutations in the gene (34).

StAR proteins are processed and imported into mitochondria, and StAR protein is induced by the secretagogues AngII and potassium in H295A adrenocortical cells (35). Previous work from our laboratory showed that: 1) bovine adrenal glomerulosa and fasciculata cells exhibited hormone-sensitive 35S-met/cys labeling of a set of eight proteins with molecular weights and isoelectric points similar to those of the hormone-sensitive rat and mouse proteins observed by Orme-Johnson (18, 19, 20, 21, 22, 23, 24, 25) and by Stocco (26, 27, 28, 29, 30, 31, 32, 33); 2) experiments with different stimuli showed that increased protein labeling and increased aldosterone synthesis both exhibited the rank order dibutyryl cAMP (dbcAMP) > AngII > potassium; 3) ANP inhibited AngII-stimulated aldosterone synthesis and AngII-stimulated alterations in the p30 proteins (36).

Our work added to the growing body of evidence that a family of 28- to 30-kDa proteins play a critical role in the acute control of a variety of steroidogenic tissues by a number of regulatory hormones. However, we had not yet shown that the set of proteins that we observed were structurally related to each other. Another unresolved issue was whether there might be species differences in the affected proteins, because our work in bovine glands had shown two sets of proteins (four of 28 kDa and four of 30 kDa), whereas others showed only four 28-kDa proteins from one 37-kDa precursor protein in rats.

One of the goals of the present study was to further define the relationship between the p30 proteins we described in bovine adrenal cells and the rat and mouse proteins described by Orme-Johnson (18, 19, 20, 21, 22, 23, 24, 25) and by Stocco (26, 27, 28, 29, 30, 31, 32, 33). In pursuit of this goal, we 1) used chymotryptic peptide mapping of the bovine adrenal glomerulosa proteins to determine if these proteins are structurally related to each other; and 2) examined labeled proteins from both bovine adrenal glomerulosa cells and rat adrenal glomerulosa cells using the same electrophoretic conditions to look for species differences. Losartan is a recently approved antihypertensive agent that blocks AngII AT1 receptors and AngII-stimulated aldosterone synthesis (37, 38, 39). Another goal of the present work was to determine the effect of AT1 blockade on p30 proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Losartan (Dup753) was a gift from Merck/DuPont (Wilmington, DE) and PD123319 was a gift from Parke-Davis/Warner-Lambert (Ann Arbor, MI). Deoxyribonuclease and ribonuclease were from Boehringer-Mannheim (Indianapolis, IN). EXPRESS protein labeling mix, containing [35S]-methionine and [35S]-cysteine 1000 Ci/mmol (35S-met/cys), and [35S]-methionine were obtained from DuPont/NEN (Boston, MA). All other reagents were obtained as previously described (36).

Preparation of cells
Bovine adrenal glomerulosa cells were prepared as described (40) and suspended in buffer containing 137 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 1.0 mM MgSO4, 11 mM glucose, 0.1% BSA, and 20 mM HEPES, pH 7.4. Rat adrenal glomerulosa cells were prepared as follows: Eight male Sprague-Dawley rats, approximately 175 g each, were killed. Adrenals were excised, placed in cold buffered saline, and trimmed of fat. Adrenal capsules were obtained by incising each gland and squeezing out the inner portion. Glomerulosa cells were prepared by collagenase digestion, as previously described (41), in a medium containing two parts DMEM with bicarbonate (Sigma catalog No. 4655; methionine-free) with one part of a modified Krebs solution. Final concentrations of electrolytes (mmol/liter) were: NaCl, 119; KCl, 3.6; MgSO4, 1.2; CaCl2, 2.54; sodium acetate, 0.4; NaH2PO4, 1.2; and NaHCO3, 17.5. The medium also contained 7.44 mmol/liter HEPES, pH 7.4; 11 mmol/liter glucose; and 1 g/liter BSA.

Incubation of cells with 35S-met/cys or 35S-methionine
Bovine adrenal glomerulosa cells were incubated in the Krebs-HEPES buffer described above, in room air, whereas rat adrenal glomerulosa cells were incubated in the DMEM described above, under 95% O2/5% CO2. Cells were preincubated for 45 min in a final vol of 1 ml at 106/ml (800,000 cells/ml for rat cells) in 15 x 75 mm nitrocellulose tubes in a 37-C shaking water bath. After preincubation, additions were made in a vol of 0.05 or 0.1 ml to provide the final concentrations indicated. These additions included AngII, ANP, dbcAMP, losartan, and PD123319, as indicated in the text and figure legends for individual experiments. High concentrations of AngII and of ANP were chosen in these experiments to help overcome any effects of ß-mercaptoethanol on AngII receptors or ANP, because this reducing agent was present in the 35S-met/cys. Immediately after the addition of these reagents, 35S-met/cys or 35S-methionine (0.1–0.2 mCi) was added to each tube and the incubation continued another 60 min, unless indicated otherwise. At the end of the incubation, tubes were chilled on ice, the contents pipetted into 1.5-ml microcentrifuge tubes, and centrifuged for one min at 10,000 x g. Cell pellets were stored at -80 C until prepared for electrophoresis. For steroid synthesis, incubations were carried out as above, except that cell concentrations were 200,000–300,000 per ml and no radioactivity was added. Aldosterone was determined in the supernatants as described (40).

Preparation of samples for electrophoresis, two-dimensional gel electrophoresis, determination of isoelectric points and molecular weights, and preparation and analysis of fluorograms were carried out as previously described (36, 42).

In situ protease digestion and peptide mapping
Bovine adrenal glomerulosa cells were incubated with 35S-met/cys in the presence or absence of 3 mM dbcAMP and proteins separated on two-dimensional gels as described previously, except that slab gels were not stained or soaked in fluorographic reagent. Instead, slab gels were dried and used to expose x-ray film immediately after electrophoresis was completed. Gels were taped securely against film and small holes punched through both before exposure. After films were developed, the punch-holes were used as an alignment guide to mark the location of proteins on the dried gel. Proteins were punched from the gel using a 3-mm cork borer, and the disks were placed into the wells of 13.5 x 14 x 0.15-cm slab gels. These gels consisted of a 4 cm-long stacking gel with 4.5% acrylamide and a 9.5 cm-long separating gel with 15% acrylamide. These gels and the running buffer were prepared according to Laemmli (43), and protease digestion was carried out according to the procedure of Cleveland et al. (44). Each sample disk was then covered with 0.07 ml of 1x stacking gel buffer containing 0.3% ß-mercaptoethanol and 10% glycerol and allowed to hydrate for 30 min at room temperature before the upper reservoir was affixed and reservoir buffer added. Immediately before electrophoresis, each well received 0.02 ml of 1x stacking gel buffer with 0.01% bromphenol blue or buffer with bromphenol blue plus chymotrypsin. Molecular weight standards also were loaded in one lane. Gels were run at approximately 20 mA per gel, carefully adjusted so that the collapsed fronts containing chymotrypsin and protein samples remained in the stacking gels for 90 min. After the samples entered the separating gels, gels were run at 60 mA until the dyefront reached the bottom of the gel. The edge with molecular weight standards was cut free and stained. The remainder of each gel was soaked in fluorographic reagent and used to expose x-ray film for approximately 2 months.

Statistical tests
Student’s unpaired two-tailed t tests were performed to obtain P values. Usually six hormonally-affected proteins were analyzed for each experiment, and more than one treatment comparison was sometimes made. For two treatment comparisons, for example, Bonferroni’s correction (45) would be 6 x 2 = 12, and to be considered statistically significant, P values should be less than (.05 ÷ 12), or P < .004. FigureGo legends provide P values, as well as the number of comparisons and the value for Bonferroni’s correction.



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  		Figure 1. Fluorograms from an experiment showing the effect of dbcAMP on incorporation of 35S-methionine into bovine adrenal glomerulosa cell proteins. Preparation and incubation of cells, two-dimensional gel electrophoresis, measurement of pH gradient, and fluorography were performed as described in Materials and Methods. Values for the pH gradient are shown underneath the middle picture. The arrows located to the right of the figure indicate the molecular weights of the two series of proteins that are affected by dbcAMP. The eight arrowheads in the fluorograms point to the locations of the proteins that exhibit different intensities in dbcAMP-treated cells vs. control cells. The top panel shows a fluorogram for control cells incubated for 60 min with 35S-methionine. The middle panel shows a fluorogram for cells incubated for 60 min with dbcAMP (3 mM) and 35S-met/cys. The bottom panel is a schematic that shows the numbers for the eight proteins which are indicated in the fluorograms by the arrowheads.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go shows the effect of dbcAMP on the distribution of 35S-labeled proteins in bovine adrenal glomerulosa cells. The upper panel shows a fluorogram for control cells and the middle panel shows one for dbcAMP-treated cells. The bottom panel provides a schematic of the eight proteins affected by dbcAMP. Proteins 1–4 are approximately 28.5 kDa, and proteins 5–8 are approximately 30 kDa. As we have previously reported, dbcAMP decreased the labeling intensity of proteins 1 and 2 and increased that of proteins 3–8, relative to control proteins.

Figure 2Go illustrates the effect of chymotrypsin digestion on proteins 1–8. Cells were originally incubated with 35Smet/cys in the presence or absence of dbcAMP. Proteins were separated by two-dimensional gel electrophoresis, and proteins 1–8 were excised from the gels and subjected to in situ chymotrypsin digestion as described in Materials and Methods. The upper panel shows a fluorogram from an experiment in which proteins 1–8 (shown in lanes 1–8, respectively) were digested with 30 ng chymotrypsin. The lane marked X shows the digestion pattern for a different, unidentified protein excised from the same 2D gel, presumably unrelated to proteins 1–8. The heavy arrow on the right indicates the position of undigested protein (protein 8) that was not treated with chymotrysin (No Enz). The small arrows to the right of the figure indicate the position of five proteolytic fragments that are common to all eight proteins, with sizes approximately 21.4, 13.1, 11.8, 11.3, and 10.4 kDa. Proteolysis products of the 28-kDa proteins (nos. 1–4) each differed in one respect from the proteolysis products of the 30-kDa proteins (nos. 5–8). Proteins 5–8 share one other fragment (approximately 24 kDa) that is not apparent or is very faint for proteins 1–4. This can be explained by retention of the structural difference that distinguishes the original 28- and 30-kDa proteins. Degradation to the 21-kDa bands occurs for all fragments, indicating removal of the distinguishing feature.



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  		Figure 2. Chymotryptic digest of affected proteins 1–8 from bovine adrenal glomerulosa cells. Cells were incubated with 35S-met/cys and dbcAMP or buffer, and proteins 1–8 were separated on two-dimensional gels, localized, excised, and subjected to in situ chymotrypsin digestion, one-dimensional electrophoresis, and fluorography, as described in Materials and Methods. The positions of molecular weight standards are shown to the left of the photographs. The upper panel depicts the results of a protease digest experiment when proteins were treated with 30 ng chymotrypsin. For comparison purposes, protein 8 was run in the lane marked No Enz, but with no chymotrypsin, and the heavy arrow to the right of the figure indicates the position of this intact protein. The lane marked X shows the chymotryptic digest of a presumably unrelated protein excised from the two-dimensional gel. The lower panel is similar to the upper panel, except that the experiment used 100 ng chymotrypsin, a different unrelated protein, and undigested protein 5 is shown for comparison.

 
The lower panel depicts an experiment in which proteins 1–8 were digested with 100 ng chymotrypsin. The proteolytic digestion patterns for proteins 1–8 were again very similar to each other. The more extensive digestion resulted in degradation to the common set of low-molecular weight fragments. Small arrows represent the fragments which had the same approximate molecular weight as the fragments indicated in the upper panel. The fact that proteins 1–8 showed very similar proteolytic fragmentation patterns strongly suggests that all eight proteins share a common primary structure.

In view of the distinctive responses of bovine adrenal p30 proteins to stimulation, the responses of rat adrenal glomerulosa cells were similarly analyzed. Figure 3Go (top panel) shows a fluorogram of a two-dimensional gel from rat adrenal glomerulosa cells, incubated with 35Smet/cys but no stimulus for 45 min. The next two panels show fluorograms from cells treated with AngII (10-7 M) and dbcAMP (3 mM), respectively. In each of the three fluorograms, arrowheads indicate the locations of proteins affected by stimuli. The bottom panel shows a schematic of these proteins. When cells were treated with dbcAMP, labeling intensity for proteins 1 and 2 decreased, whereas that for proteins 3 and 4 increased. AngII treatment also slightly increased labeling of proteins 3 and 4. Proteins 1–4 were approximately 28.4 kDa, very similar to the bovine proteins 1–4, which are 28.5 kDa. Isoelectric points for rat glomerulosa proteins 1–4 were approximately 6.8, 6.7, 6.6, and 6.5, similar to the hormonally affected rat adrenal fasciculata proteins observed by Orme-Johnson’s group. dbcAMP also increased labeling for one other protein (not numbered, but indicated by an arrowhead in the schematic and in the gel photograph). This protein was approximately 35 kDa, with an isoelectric point of 7.0, and thus is similar to the pp37 protein (37 kDa, isoelectric point 7.1) stimulated by dbcAMP in rat fasciculata cells (24). There was no indication of stimulation of a set of 30-kDa proteins. In this experiment, both stimuli increased aldosterone synthesis (25.4 ± 1.2 vs. 2.9 ± 0.2 vs. 0.3 ± 0.1 ng aldosterone/106 cells/h for dbcAMP vs. AngII vs. control).



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  		Figure 3. Effect of AngII and dbcAMP on 35S-met/cys incorporation into rat adrenal glomerulosa cells. Cells were incubated for 45 min with 35S-met/cys in the presence or absence of AngII or dbcAMP, as shown in the figure. This experiment included three control incubation tubes, two with dbcAMP, and two with AngII. Proteins were analyzed by two-dimensional gel electrophoresis and fluorography, as described in the text. Values for the pH gradient are shown underneath the bottom gel photographs. The heavy arrow located to the right of the gel photographs indicates the molecular weight of the series of four proteins that are affected by dbcAMP. Arrowheads in the fluorograms, and in the schematic, point to the locations of this series of four proteins. One other arrowhead indicates the location of one other protein (35.3 kDa, pI approximately 7.0) which exhibited increased labeling in the presence of dbcAMP. When films from both dbcAMP incubations were examined, in comparison with all three control incubations, dbcAMP in each instance decreased labeling intensity of proteins 1 and 2, increased that of proteins 3 and 4, and increased that of the 35.3-kDa protein.

 
Previous work from this laboratory with bovine adrenal glomerulosa cells has shown that AngII decreased labeling of proteins 1 and 2 and increased labeling of proteins 3–8, but that ANP blunted these effects of AngII on these proteins while inhibiting aldosterone synthesis (36). These changes are confirmed by the data in Fig. 4Go. The effects of AngII were much less than those of dbcAMP, and appearance of proteins 7 and 8 was often undetectable. Changes in proteins 5 and 6 provided the best indicator of an AngII effect. Figure 4Go shows the results of an experiment testing the effects of losartan, the AT1 receptor antagonist, and ANP. ANP blunted AngII’s effect on proteins 1–4 and, most evidently, on 5 and 6. Losartan blunted the effects of AngII on these proteins more effectively than ANP. The lower panel shows the results of this experiment, analyzed by densitometry. Losartan decreased AngII-stimulated changes in the six proteins, so that the pattern seen for AngII plus losartan treatment was similar to that for control cells. ANP produced an intermediate effect.



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  		Figure 4. Upper panel, Fluorograms from control cells (C) and cells incubated with 35S-methionine and with AngII (AngII), AngII plus ANP (AngII+ANP), or AngII plus losartan (AngII+Los). The fluorogram for control cells is seen in the top panel. The second, third, and fourth panels show fluorograms for cells incubated with AngII (300 nM), AngII (300 nM) plus ANP (500 nM), or AngII (300 nM) plus losartan (100 µM). Values for the pH gradient are shown below the bottom fluorogram. The arrows located to the right of the figure indicate the molecular weights of the two series of proteins that are affected by treatment. The six arrowheads in the fluorograms point to the locations of the proteins that are affected by hormonal treatments. Lower panel, Effects of AngII, ANP, and losartan on protein labeling for this experiment, as analyzed by densitometry. Spot density values are shown, to provide a quantitative comparison among proteins 1–6 for the four treatment groups. The effects of AngII on proteins 7 and 8 were too faint for densitometric analysis. Fluorograms of two-dimensional gels were analyzed by the Collage System, and spot density values were normalized by use of the benchmark proteins, as described previously (36). For each of the proteins, spot density was then expressed as a percentage of the spot density for that protein from control cells. For cells treated with AngII or with AngII plus losartan, cell incubations were carried out in duplicate, so that the mean and range of the two values are shown for these treatment groups. The following P values were obtained in comparing the AngII treatment vs. AngII plus losartan: For protein 1, P = .070; for protein 2, P = .089; for protein 3, P = .0053; for protein 4, P = .011; for protein 5, P = .006; and for protein 6, P = .060. Because of the multiple comparisons being made (six proteins), the Bonferroni correction would recommend that P should be less than .008, to be statistically significant at the 5% level.

 
In other experiments, the effects of selective antagonists for the AT1 and AngII type 2 (AT2) receptors were studied in bovine adrenal glomerulosa cells. Figure 5Go shows the aldosterone synthesis response when cells were incubated with AngII in the presence and absence of losartan (an antagonist of the AT1 subtype) or PD12339 (an antagonist of the AT2 subtype). AngII stimulated aldosterone synthesis approximately 4-fold, and losartan (but not PD 123319) completely antagonized AngII’s effect. Figure 6Go shows the protein labeling data from the same batch of cells. AngII decreased the labeling intensity of proteins 1 and 2 and increased that of proteins 3–6. Losartan inhibited AngII’s effects on all six proteins, restoring protein labeling values toward those for control cells. The higher concentration of losartan (10-5 M) was more effective than the lower dose (10-6 M). PD123319, on the other hand, was not as effective as losartan in blocking AngII’s effects. In another experiment, losartan again blocked AngII-stimulated changes in protein labeling, but PD123319 had no effect.



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  		Figure 5. Aldosterone synthesis from control bovine adrenal glomerulosa cells, or cells incubated with AngII, AngII plus losartan, or AngII plus PD123319. Cells were incubated at the same time as the cells incubated with 35S-methionine for the experiment depicted in Fig. 6Go. Aldosterone was determined in the cell supernatants as described in the text and is expressed as ng aldosterone produced per 106 cells per h. Each bar represents the mean ± SE from four incubations tubes. The following P values were obtained: AngII vs. control, P < 0.001; AngII plus 10 uM losartan vs. AngII alone, P < 0.001; AngII plus PD123319 vs. AngII alone, P = 0.65 (no significant difference).

 


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  		Figure 6. Effects of AngII and losartan on protein labeling for the experiment depicted in Fig. 5Go. Similar results were obtained for AngII, losartan, and PD123319 in another independent experiment. Spot density values show the quantitative comparison among proteins 1–6 for the four treatment groups. The effects of AngII on proteins 7 and 8 were too faint for densitometric analysis. For each protein, spot density was expressed as a percentage of the spot density for that protein from control cells. Fluorography and measurement and normalization of spot densities were as described previously (36). Bars represent the mean ± SE from cell incubations carried out in triplicate in the experiment. The following P values less than 0.05 are as follows: For control vs. AngII-treated cells: for protein 2, P = .021; for protein 3, P = .013; for protein 4, P = .0002; for protein 5, P = .028; and for protein 6, P = .00002. For cells treated with AngII vs. AngII plus 1 uM losartan: for protein 1, P = .014; for protein 2, P = .010; for protein 3, P = .031; for protein 4, P = .004; and for protein 6, P = .0002. For cells treated with AngII vs. AngII plus 10 uM losartan: for protein 1, P = .007; for protein 3, P = .009; for protein 4, P = .012; for protein 5, P = .006; and for protein 6, P = .0001. For cells treated with AngII vs. AngII plus 10 uM PD123319: for protein 4, P = .005; for protein 6, P = .006. Because of the multiple comparisons being made (six proteins), the Bonferroni correction would recommend that P should be less than .002, to be statistically significant at the 5% level.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is now a substantial body of evidence that a family of 28- to 30-kDa proteins (p30 proteins or StAR proteins) is involved in hormonal stimulation in a variety of steroidogenic tissues in several species.

Previous work from this laboratory showed that secretagogues affect several proteins in bovine adrenal cells (36). dbcAMP decreased the labeling of two proteins (nos. 1 and 2) and increased that of six proteins (nos. 3–8) in bovine adrenal glomerulosa and fasciculata cells. We pointed out the similarity between proteins 1–4 in bovine adrenal glomerulosa cells and proteins p30, p30', pp30, and pp30', respectively, in rat adrenal fasciculata cells observed by Orme-Johnson (20, 24), similarities based on molecular weights, isoelectric points, and hormone responsiveness. The p30 proteins described by others also had been shown by protease mapping to share much primary structure. Orme-Johnson suggested that a set of four peptides could be produced from a core peptide (p30) and combinations of two different modifications (to produce pp30, p30', and pp30') (20, 24).

The present work, using chymotryptic digests, shows that all eight bovine adrenal glomerulosa proteins exhibit similar fragmentation patterns, consistent with the proteins sharing a common primary structure. However, there was no obvious analogy between our bovine proteins 5–8 and rat adrenal proteins. The 30-kDa proteins 5–8 may include an additional peptide sequence not shared by the 28.5-kDa proteins 1–4 and not present in rat. Chymotryptic digest patterns in the present report are consistent with the 30-kDa proteins possessing some additional amino acid residues at one end of the protein that are retained after cleavage at one terminus (producing a 24-kDa fragment) but not after cleavage at the other terminus (producing a 21-kDa fragment). It is possible that the sets of four 30-kDa and four 28.5-kDa proteins arise from combinations of two distinct modifications of both the 30-kDa and the 28-kDa core proteins.

A summary of our observations and ideas about the protein structures is as follows: 1) Rat cells and bovine cells have a core 28.5-kDa protein that can undergo combinations of two distinct hormonally stimulated modifications to produce four 28.5-kDa proteins; 2) hormonal treatment of bovine cells, but not rat cells, stimulates production of a 30-kDa precursor protein that is structurally similar to the 28.5-kDa protein; and 3) modification of the bovine 30-kDa protein is similar to that of the 28.5-kDa protein and leads to four 30-kDa proteins.

Although there are species differences in the exact proteins seen in response to hormonal stimulation, these differences are overshadowed by the great similarities in overall characteristics seen between different species and tissues for the p30 or StAR proteins. These similarities include molecular weight, isoelectric points, responsiveness to hormones that are stimulatory or inhibitory for different steroidogenic tissues and hormones, and the fact that within each group of proteins, much primary structure is shared.

Stimulation of aldosterone synthesis by AngII is mediated by AT1 receptors, whereas AT2 receptors play no apparent role in aldosterone synthesis (39). We have shown that AngII increases aldosterone synthesis, decreases the labeling of StAR proteins 1 and 2, and increases that of proteins 3–6. Losartan, a competitive antagonist of the AT1 receptor, not only inhibited AngII-stimulated aldosterone synthesis, as previously shown by others (39), but also inhibited AngII’s effects on proteins 1–6 in bovine cells. PD123319, which specifically antagonizes the binding of AngII to the AT2 receptor, did not affect AngII-stimulated aldosterone synthesis and had little effect on AngII-induced changes in protein labeling. PD123319 inhibited labeling of proteins 4 and 6 to a small extent in one experiment and had no effect in another experiment. Possible interpretations of these results are that: 1) AngII’s effects on proteins 4 and 6 are not necessary for aldosterone stimulation of aldosterone synthesis; or 2) PD123319’s effects on proteins 4 and 6 are too weak to inhibit aldosterone synthesis; or 3) the observed effect of PD123319 on proteins 4 and 6 was caused by chance or experimental error and, in fact, PD123319 has no effect on proteins 4 and 6. Further experimentation is necessary to clarify this issue. Our results clearly indicate that the effects of AngII on proteins 1–6 are inhibited by blocking the AT1 receptor with losartan.

The effects of losartan on AngII-stimulated aldosterone synthesis and on proteins 1–6 are very similar to those of ANP. ANP does not block AngII receptors but antagonizes AngII’s effects at some point further downstream in signal transduction by a poorly understood mechanism (14). The fact that two agents that inhibit AngII-stimulated aldosterone synthesis in different ways both inhibit AngII’s effects on proteins 1–6 underscores the importance of these proteins in control of aldosterone synthesis.

The observation that AngII, potassium, and dbcAMP affect aldosterone synthesis and p30 proteins similarly (although to different extents), and that ANP antagonizes the effects of AngII on both aldosterone synthesis and p30 proteins, suggests that these proteins mediate the acute effects of stimuli on aldosteronogenesis. The present demonstration that AT1 receptor blockade inhibits both AngII-stimulated aldosterone synthesis and AngII’s effects on the StAR proteins provides additional evidence that these proteins play a role in the acute control of the adrenal glomerulosa by the renin-angiotensin system.


   Acknowledgments
 
We thank Brad Haley and Prof. Neal First for acquisition of bovine adrenal glands; and Kathryn Kleckner, Jeffrey Root, and Teresa Sacia for illustrations and photography. We thank Dr. Ellen Roecker, of the University of Wisconsin Biostatistics Department, for advice on statistical analysis of the data. We are grateful to Prof. N.R. Orme-Johnson for helpful discussion. We thank Prof. Robert Bremel, of the University of Wisconsin-Madison Animal Sciences Department, for the use of the Collage Image Information Extraction and Reduction System.


   Footnotes
 
1 This work was supported by NIH Grant DK-18585 (to C.R.J.), by a grant from Merck/DuPont, and by the Department of Veterans Affairs. Back

Received October 25, 1996.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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